In order to detect and quantify DNA methylation in sBCC samples 10-14, Infinium MethylationEPIC BeadChips (Illumina, San Diego, USA) with over 850’000 methylation sites were used in combination with bisulfite conversion of DNA samples and the Infinium HD Assay for Methylation. First, gDNA was modified by bisulfite conversion. Using the EZ DNA MethylationTM Kit (Zymo Research, Irvine, USA), 500 ng of methylated DNA was treated with bisulfite according to manufacturer’s instructions, thereby converting unmethylated cytosine into uracil. After the on-column desulphonation step, bisulfite converted DNA samples were then further processed using the Illumina Infinium HD Assay for Methylation (Illumina, San Diego, USA) according to manufacturer’s instructions: First, bisulfite converted DNA was denatured and neutralized. Second, the DNA was amplified by three orders of magnitude in a whole-genome amplification step. After enzymatic fragmentation, the amplified DNA was precipitated using isopropanol. After resuspension, the DNA was hybridized onto the microarrays. The microarray hybridization was performed according to the Infinium HD Assay Methylation Protocol Guide (Illumina, San Diego, USA). The bisulfite-converted DNA samples were hybridized at 48°C for 16 hrs on separate Illumina Infinium MethylationEPIC Arrays. DNA fragments anneal to locus-specific 50 nucleotide oligomers which are covalently linked to different bead types.
Label
Cy3 and Cy5
Label protocol
Allele-specific single base extension of the primer incorporates a biotin nucleotide or a dinitrophenyl labelled nucleotide (C and G nucleotides are biotin labelled, A and T nucleotides are dinitrophenyl labelled). Hybridized DNA was then removed and the labelled extended primers were stained and dried.
Hybridization protocol
After hybridization and washing of the microarrays, the primers on the BeadChips were extended complementary to the binding DNA sample. This reaction incorporates labelled nucleotides. Incorporation of a G corresponds to the methylated state, A to the unmethylated state on the complementary strand.
Scan protocol
Fluorescent signal intensities of the labelled and stained nucleotides on the BeadChips were detected with a dual color channel approach and the Illumina iScan™ System (Illumina, San Diego, USA).
Data processing
Intensity files were generated by the iScan System for the green and the red channel (Illumina, San Diego, USA). The software tool GenomeStudio V2011.1 (Illumina, San Diego, USA) was used for quality control and visualization of the performance of microarray analysis as well as for methylation analysis. Raw microarray data were imported into GenomeStudio V2011.1 and analyzed. For the Infinium Methylation Assay, the β value was calculated as: β = Max (Signal B, 0) / Max (Signal A, 0) + Max (Signal B, 0) + 100. For Infinium I assays, signal A and signal B were produced by two different bead types and reported in the same color. The file Methylation_Profiling_Supplementary_Material_II.xlsx includes both signal intensity and average Beta values.
Submission date
Mar 25, 2019
Last update date
Oct 27, 2019
Contact name
Michael Sand
Organization name
Ruhr-University Bochum
Department
Depatment of Dermatology, Venereology and Allergology
